PROJECT SUMMARY Lower urinary tract symptoms (LUTS) affect millions of people and are especially prevalent in the elderly population. LUTS are likely caused or exacerbated by dysfunction of neural circuits controlling bladder function. Despite some progress in our understanding of the circuits that control reflex and voluntary micturition, significant knowledge gaps remain. An enhanced understanding of how finely tuned and effective neural control over bladder function is achieved is central to efforts directed at developing newer and more targeted treatments for LUTS. The objective in this particular application is to understand which neurons detect, relay and process the bladder distention signal, so that it ultimately becomes integrated into coherent neural control for proper bladder function. The central hypothesis is that periaqueductal gray (PAG) neurons that receive bladder fullness information (PAG?sense?) ?gate? neurons in the pontine micturition center (PMC) to become activated to initiate micturition behavior. Successful bladder filling and voiding is directed by PMC neurons that project to spinal cord motoneurons that, in turn, innervate detrusor and urethral sphincter muscles. Our model predicts that activity in PMC neurons is likely suppressed until the sensory (bladder distention) signal has been relayed/distributed (from spinal cord-efferent PAG neurons) to brain regions that exert ?executive control? in determining whether a situation is safe and socially acceptable for voiding. The proper function of both first-pass sensing neurons and of these inputs, including those from the PAG, that exert inhibitory control over PMC neuron activity are critical for maintaining continence. Guided by strong preliminary data, we will test our overarching hypothesis by pursuing three specific aims: 1) identify and map axonal projections of sacral spinal cord-efferent PAG/PAG?sense? neurons; 2) using fiber photometry Ca2+ imaging, define neural activity in bladder-afferent- activity recipient PAG/PAG?sense? neurons that may function to transform the distention signal to PMC output action, and determine how inhibitory PMC-afferent neurons, activate or deactivate to allow voiding; and 3) using optogenetic stimulation define the circuit basis of inhibitory neural control over bladder function, including identifying neurons that are necessary for maintaining continence. The approach is intellectually and technically innovative because of its emphasis on sensory-signal-sensing PAG neurons and on inhibitory afferent inputs (and source cell populations) that regulate PMC activity, and because it employs a novel combination of newly developed and validated technical approaches. This work is significant because it is one of several key steps in a continuum of research that is expected to lead to significant improvement of knowledge of our understanding of the cellular and synaptic circuits that control bladder filling and voiding. Collectively these studies will inform a far deeper and more detailed understanding of the circuitry regulating bladder function and hence has the very real potential to inform the development of newer therapeutics for treating LUTS.